Candidate: The candidate is a board-certified veterinary surgeon with extensive research training in animal models and basic science who transitioned after earning a PhD in gastrointestinal physiology to postdoctoral training in biomedical engineering. Her immediate career development goals in the current proposal will be in biomechanics and tissue engineering and will allow expansion of current skills in mechanical testing and stress-strain analysis, biomaterial development, confocal microscopy, image and data post-processing and fast Fourier transform analysis, skills essential to the success of the research project and long term career goals as an independent investigator in the field of tendon and ligament tissue engineering. The candidate plans R03 submission in year 2 to evaluate biocompatibility of the proposed scaffolds in rodent models, and then an R01 submission in year 4 to evaluate the proposed scaffolds in a large animal model of chronic rotator cuff tear, and expects to publish three manuscripts resulting from this initial work. Additional didactic training in tissue engineering, statistics and responsible conduct of research will be obtained during the award period. Environment: The mentorship of Dr. Farshid Guilak PhD and co-mentorship of Dr. David Ruch MD, combined with the degree of institutional commitment will provide this candidate with the support needed to transition successfully into an independent research career. External consultation from experts in rotator cuff disease, research and repair will provide added expertise. All the equipment and facilities needed by the candidate for successful completion of this proposal are already available in the Orthopaedic Research Laboratories in the Department of Surgery, and the candidate has 100% protected time, with no administrative, clinical or teaching responsibilities to allow for the successful completion of this project. Research: The rotator cuff is a composite of joint capsule, tendons and ligaments that is critical for stability and function of the shoulder. There are over 300,000 rotator cuff injuries repaired annually in the US, at an annual burden in surgical expenses alone of $3-4 billion. Suture repair is the clinical standard of care but re- tear rates of 34-94% have been reported. Extracellular matrix (ECM) scaffolds are used for augmentation of rotator cuff repair because they contain bioactive molecules that stimulate cell migration, proliferation and ECM synthesis. All of the currently available ECM scaffold patches used to augment suture repair have limitations and none mimic the interdigitating multi-layered structure of the normal rotator cuff which has layers of collagen aligned at 45 degrees to each other to allow efficient load transfer and distribution, suture retention and resistance to failure. Therefore, there is a need for a rotator cuff patch that meets all of the deficiencies of currently available devices, while retaining the benefits of using ECM. The long term goal of the proposed research is to develop a tendon or ligament derived-extracellular matrix (TLDM) patch for augmentation of rotator cuff repair or as a tendon substitute in irreparable rotator cuff tears while allowing the PI to establish an independent research career in tissue engineering of tendon and ligament. Electrospun nanofibers are attractive for tissue engineering of a rotator cuff patch because the fibers can be aligned to mimic the natural architecture and biomechanical properties of native tissue. Electrospinning into an aqueous solution and collection of multiple layers of scaffold (hydrospinning) increases porosity and further enhances cell infiltration compared to more traditional nanoscaffolds deposited as a single layer onto a solid ground plate. We have shown that a novel TLDM enhances proliferation and early differentiation of human adipose stem cells (hASCs) towards a tendon phenotype and that TLDM can be used to produce hydrospun nanofiber scaffolds. We have developed a novel strategy to align adjacent layers of hydrospun nanofibers in different directions by modification of the shape of the electric field. The PI is well prepared to meet the scientific and training goals of this application through the research experience already acquired in the proposed research and mentorship environment, and through the mentorship and continued development in this environment covered in this proposal. The overall hypothesis is that aligned TLDM scaffolds will show anisotropic ECM maturation compared to non-aligned scaffolds, and scaffolds with layers aligned at 45 degrees to each other (45-Offset) will demonstrate increased mechanical properties over a range of load directions than TLDM scaffolds with all layers aligned similarly (0- Offset) or non-aligned TLDM scaffolds. Use of 45-Offset layers of hydrospun fibers represents an innovative approach to promote early ECM synthesis, alignment and maturation in a multi-layered RC tendon TLDM patch. We will test the following hypotheses: Hypothesis 1: Scaffold fiber alignment and mechanical anisotropy will be greatest in 0-Offset scaffolds followed by 45-Offset scaffolds and finally non-aligned TLDM scaffolds. Hypothesis 2: 45-Offset and 0-Offset scaffolds will show increased cell and ECM alignment and maturation when seeded with hASCs, compared to non-aligned scaffolds. We expect that 45-Offset aligned TLDM scaffolds will permit earlier and more aligned ECM synthesis and maturation compared to non-aligned scaffolds and reduced mechanical anisotropy compared to 0-Offset aligned scaffolds.